This invention relates to implantable biocompatible compositions that induce the repair of damaged or diseased bone, cartilage or other connective tissues upon contact of the damaged or diseased tissues with the composition in vivo. The invention also relates methods of inducing repair. More particularly the present invention is directed to the use of a composition comprising an effective amount of pleiotrophin to induce repair of damaged or diseased connective tissues.
Currently, bone defects are typically repaired by autografts or banked bone. Autografts have a good ability to unify the bone, and physicians often prefer to use bone from sources such as the iliac crest. However, procedures using autografts suffer from several drawbacks. First, autografts require a separate harvest operation, resulting in increased operative time and the use of blood transfusions. Secondly, patients often lack adequate amounts of material for harvesting and often experience donation site morbidity. Implantation of banked bone does not require the harvest operation, but its bone healing capability is not as high as that of autografts. Therefore, it is undesirable to use banked bone in severe conditions such as nonunion.
Because of these drawbacks, researchers have searched for compositions and methods for promoting bone growth without necessitating the use of autografts or banked bones. One potential source for bone growth promoting factors is the extracellular matrices of healthy bone and cartilage tissues. Extracellular bone matrix contains predominantly mineral (hydroxyapatite) and an organic matrix, where the major component of the organic matrix is collagen type I. The remaining components of bone matrices include a number of less abundant non-collagenous proteins and growth factors. For example, since the mid-1960""s the osteoinductive activity of both demineralized bone matrix (DBM) and bone morphogenetic protein (BMP) has been studied (Ijiri, 1992). In addition to DBM and BMP, many non-collagenous bone matrix protein components possess biological activity and find wide use in medical applications such as prosthetic devices, drugs, blood components, and the like.
One non-collagenous protein found in bone matrix is pleiotrophin (also known as HB-GAM, HARP, P18, and OSF-1). Pleiotrophin is a heparin-binding protein belonging to a recently described family of developmentally regulated cytokines. Pleiotrophin has been reported to exhibit both neurite outgrowth-promoting activity, on embryonic rat brain neurons in culture, and mitogenic activity toward rat and mouse fibroblasts. The biological activity of recombinant human pleiotrophin is measured by its ability to enhance neurite outgrowth of cerebral cortical neurons of E10 chicken embryos. The EC50 is defined as the effective concentration of growth factor that elicits a 50% increase of neurite outgrowth from E10 chicken embryos.
Pleiotrophin protein was initially isolated from early postnatal rat brain and bovine uterus, and can be isolated from warm-blooded vertebrate bone and cartilage tissues using standard extraction techniques known to those skilled in the art, including extracting with guanidine hydrochloride. Human pleiotrophin protein is initially produced as a 168 amino acid residue precursor that is processed to remove the 32 amino acid residue signal peptide and produce the 136 amino acid mature protein. DNA sequences encoding the protein have been recently cloned and recombinant pleiotrophin protein is commercially available from Sigma chemical company (St. Louis, Mo.). Gene sequences (cDNA clones) encoding for pleiotrophin have been prepared from rat, bovine and human cell mRNAs. The predicted amino acid sequence of pleiotrophin is highly conserved between these three species, and each encoded protein contains a signal sequence, suggesting that the protein is secreted.
Pleiotrophin binds strongly to bone extracellular matrix in vitro and may have an autocrine or paracrine action. Pleiotrophin shares some chemical similarities with the family of fibroblast growth factors (FGF), namely the heparin-binding ability, however pleiotrophin does not belong chemically to the FGF family. The deduced amino acid sequence of pleiotrophin is distinct from any other known growth factor family. Pleiotrophin is known to be expressed by osteoblasts, cartilage and osteosarcoma cells, and has been isolated from bone and cartilage. The expression of pleiotrophin has been associated with bone and cartilage progenitor cells, as well the mesenchyme of lung, gut, kidney, and reproductive tract of developing rat tissues.
Numerous compounds have been isolated from biological sources and have been identified as having potential bone growth enhancing activities based on the response of cultured cells to those compounds. In particular, due to the association of pleiotrophin with developing bone in animal tissues, and the response of various cultured cells to pleiotrophin in vitro, it has been suggested that this molecule may play a role in bone and/or cartilage development. However, in vitro results may vary due to a variety of factors, including cell type, cell density, cell isolation procedures, and type of growth medium. Therefore, while useful, in vitro studies are not always predictive of in vivo activity.
For example over the last two decades prostaglandins had been reported as both increasing bone resorption as well as increasing bone formation. Analysis of the literature references reporting the conflicting activities of prostaglandins reveals that almost all reports of bone resorption were performed in vitro and almost all the studies reporting bone formation were done in vivo (Mark and Miller, 1993). Previous studies of bone growth in vitro were performed with tissue/organ cultures of bone or relatively pure isolated bone cell populations. The apparent conflicting reports of the predominant skeletal affects of the prostaglandins can be explained on the basis of the limitations of the cell culture systems used to study those effects. Similarly, initial reports of TGF- activity based on cell culture assays failed to correlate with observed in vivo activities. Therefore, skilled artisans appreciate that in vitro activity does not always predict in vivo results.
What is needed are compositions shown to repair connective tissue in vivo.
In accordance with the present invention, compositions comprising pleiotrophin are used to induce growth of bone or cartilage at an in vivo site in need of repair. The disclosed composition is administered to a warm-blooded species, either by implanting or injecting the composition, for in vivo contact with the site in need of repair. This invention is the first demonstration that pleiotrophin alone is capable of enhancing the repair of bone or cartilage tissues by endogenous tissues.
Another aspect of this invention is a method for inducing new bone or cartilage growth at a predetermined in vivo site of a vertebrate species comprising the steps of contacting the site with a composition comprising substantially purified pleiotrophin, in an amount effective to induce endogenous tissue growth, and a pharmaceutically acceptable carrier. In a preferred embodiment, the composition is in liquid form and the site is contacted by injection of the composition. In another preferred embodiment, the carrier is a polymer matrix comprising a polymer selected from the group consisting of polyesters, ionomers, poly(amino acids), polyvinyl acetate, polyacrylates, polyorthoesters, polyanhydrides, collagens, fibrins, starches, alginate, and hyaluronic acid. Alternatively, the carrier may be a metal, glass, or mineral salt. Preferred mineral salts include tricalcium phosphate, hydroxyapatite, and gypsum.
Still another aspect of this invention is a method of treating a bone or cartilage pathogenic condition in a warm-blooded vertebrate by administering a composition systemically to the warm-blooded vertebrate, wherein the composition comprises substantially purified pleiotrophin, in an amount effective to induce endogenous tissue growth, and a pharmaceutically acceptable carrier.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of embodiments including those exemplifying the best mode of carrying out the invention.
The present invention is directed to compositions comprising pleiotrophin in a substantially pure form, and the use of such compositions to enhance the repair of bone and cartilage defects in vivo. As used herein the term xe2x80x9cpleiotrophinxe2x80x9d is intended to include native pleiotrophin protein isolated from human or other warm-blooded vertebrates, recombinant protein produced from pleiotrophin encoding nucleic acid sequences, and protein fragments/peptides of pleiotrophin proteins. A pleiotrophin gene is defined herein to include any nucleic acid sequence encoding for pleiotrophin, including the native gene sequences isolated from human or other warm-blooded vertebrates, any nucleic acid sequence encoding active fragments of pleiotrophin, or any recombinant derivative thereof. As used herein, the term substantially pure is intended to mean purified to at least 90% pure, and preferably to 95% purity, as determined by polyacrylamide gel electrophoresis or amino acid analysis.
The compositions of the present invention can be used in a method for inducing the repair of damaged or defective tissues of a warm-blooded vertebrate. More particularly, pleiotrophin can be used to repair the tissues of orthopedic and non-orthopedic wound sites, including bone, cartilage, tendon, ligament, muscle, skin, and other soft tissues. In one embodiment the compositions of the present invention are used to repair fractures effectively and fill or bridge bone defects including for example, craniofacial defects or periodontal defects, joint fractures, chondral defects, superficial chondral defects, full thickness defects, osteochondritis dissecans, minuscule tears, ligament tears, tendon tears, muscle lesions, myotendinitis junction lesions, skeletal reconstruction following secondary bone loss to infection or neoplasm, and the treatment of various bone or cartilaginous diseases resulting in defects.
Many compounds that have been isolated from connective tissues have been reported as having osteogenic properties based on the response of cells to those compounds in vitro. However, in vitro-established activities often fail to provide sufficient guidance for selecting compounds that will exhibit the desired in vivo bone and cartilage repair enhancing activity. Accordingly, the present invention uses an in vivo assay technique to identify bioactive agents that induce the repair of bone and cartilage tissues. Advantageously, the present in vivo assay avoids the use of delivery carriers, such as collagen, which are known to exhibit osteogenic properties themselves.
This unique in vivo assay was used to identify compounds that enhance bone repair. More particularly, the in vivo assay described in Example 1 demonstrates that pleiotrophin enhances the repair of bone in vivo.
Pleiotrophin may enhance the repair of these tissues either directly or indirectly. For example, pleiotrophin may increase new bone formation at a localized site by directly stimulating osteoblast activity (i.e. by enhancing matrix production or by recruiting additional osteoblast cells), by increasing angiogenesis, or by inhibiting osteoclast resorption. In addition, the compositions of the present invention may participate in the recruitment of bone progenitor cells or bioactive agents to the localized site either by selective binding of pleiotrophin to the progenitor cells or the bioactive agent, or pleiotrophin may participate in the recruitment of cells through chemotaxis. It is also anticipated that pleiotrophin can be used in a wound repair context in combination with a carrier material such as a ceramic or polymer, including the use of proteins such as collagen as the carrier material. In addition pleiotrophin can be combined with autologous cells (such as bone or cartilage progenitor cells) or autologous proteins (such as fibrin).
In an embodiment the compositions of the present invention comprise a delivery vehicle and a bioactive mixture comprising an effective amount of a substantially pure pleiotrophin. In another embodiment, the compositions of the present invention comprise a delivery vehicle and a bioactive mixture comprising a pleiotrophin gene. Delivery vehicles suitable for use in delivering bioactive agents to bone and cartilage in vivo are well known to those skilled in the art. In one embodiment the delivery vehicle comprises a polymer matrix, and the polymer matrix is formed from one or more biocompatible polymers. As used herein, xe2x80x9cbiocompatiblexe2x80x9d means that the polymer is non-toxic, non-mutagenic, and elicits a minimal to moderate inflammatory reaction. Preferably the biocompatible polymer is also biodegradable and completely degrades in a controlled manner into non-toxic residues. In this embodiment, the polymer matrix serves as a delivery vehicle for the bioactive mixture, concentrating the bioactive agent at a localized site of administration and controlling the release of the bioactive composition. The controlled delivery and release of pleiotrophin to localized bone and cartilage sites is based on the use of biodegradable, biocompatible polymers in combination with bioactive molecules to achieve both efficacious release of molecules and removal of the polymer from the treatment site within a physiologically useful time period.
A variety of polymers can be used to form the implant for the purposes of delivering bioactive molecules to a predetermined in vivo site, including polyesters, polyvinyl acetate, polyacrylates, polyorthoesters, polyhydroxyethylmethacrylate (polyhema) and polyanhydrides. One of the advantages of polyesters in such applications is that they are both biodegradable and biocompatible. Aliphatic polyesters have been widely used in the area of biomaterials for implantable drug delivery devices, sutures, and general tissues supports, after injury or surgery. The polyesters traditionally of greatest interest for localized delivery of compounds, are derived from lactide, glycolide, and -caprolactone monomers, with a fairly broad range of degradation profiles accessible through various termonomer combinations. The ester linkages in these aliphatic polyesters are hydrolytically and/or enzymatically labile and render the polymers degradable in aqueous environments.
In a preferred embodiment, polymers such as polyanhydrides, polyester anhydrides, or ionomers are used. Alternatively, other polymers such as polylactic acid and polyorthoesters can also be used. In another embodiment the polymer matrix comprises collagen fibers. Collagen has been reported to exhibit bioactive properties and enhances the repair of bone and cartilage tissues in vivo. Accordingly collagen fiber can function as both a component of the delivery system as well as an active agent of the present bone and cartilage repair compositions. Other polymers suitable for use in forming the polymer matrix comprise fibrins, starches, alginate, and hyaluronic acid.
The composition of the polymer used to form the delivery vehicle matrix, as well as the molecular weight and physical properties of the polymer, can be varied according to the application. For example, hydrophobic polyanhydrides can be used where it is desirable to increase the time of degradation. Compounds can be mixed into, or polymerized with the polymer as required for additional strength or other desirable physical properties, using materials known to those skilled in the art of polymer chemistry or materials science. For example, tricalcium phosphate or hydroxyapatite particles that provide better physical handling properties can be added to the composition.
In general, for repair of bone breaks, the polymer should release the material over a period of approximately 3 to 42 days (generally six weeks are required for sufficient repair to occur in humans before the bone is capable of bearing weight). The polymer should also degrade completely over a period no longer than about sixteen to twenty weeks. Release and degradation times will depend in part upon the polymer used and the bioactive materials to be released. In addition to polymers, various other time-release vehicles are known. An approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a more or less constant rate of drug release is maintained. See, e.g., U.S. Pat. No. 3,710,795. Another approach is the use of an osmotic pump, as described in Example 1.
In accordance with an embodiment of the present invention, the delivery vehicle comprises polyester ionomers (salts of carboxy-terminated polyesters). The polyester ionomers exhibit good solubility even at higher molecular weights dictated by implant structural/functional requirements. The polyesters are prepared from and degrade into naturally occurring metabolites for enhanced biocompatibility. The polyester ionomers are prepared from the corresponding carboxy-terminated polyesters by neutralization or partial neutralization with biocompatible, pharmaceutically acceptable salt-forming bases. In an embodiment the delivery vehicle comprises biodegradable carboxy-terminated polyesters in combination with the corresponding ionomers. The physical properties of polyester ionomers can be controlled by the degree of neutralization of the corresponding carboxyterminated polyesters and to some extent by selection of the neutralizing base. The polyester ionomers can be used alone or in combination with their carboxy-terminated polyester precursor for use in construction of a biocompatible delivery vehicle for tissue repair and/or prolonged release of biologically active compounds.
The use of polyester ionomers as delivery vehicles is described in U.S. Pat. No. 5,668,288, the disclosure of which is incorporated herein by reference. In general the polyester ionomers are divalent residues of a polyester. The polyester can comprise a homopolymer, copolymer, or terpolymer of biocompatible hydroxy acids, for example, lactic acid, glycolic acid, -hydroxy caproic acid, and -hydroxy valeric acid. Alternatively, the polyester can be formed using copolymerization of a polyhydric alcohol and a biocompatible polycarboxylic acid. Most typically such copolymers are formed between dihydric alcohols, for example, propylene glycol for biocompatibility and biocompatible dicarboxylic acids.
The bioactive component of the present compositions comprises pleiotrophin optionally combined with a pharmaceutically acceptable carrier, solubilizing agent, or filler material. To induce bone growth formation, pleiotrophin should be administered at a concentration ranging from about 50 ng to about 10 mg/ml of the defect area. In an embodiment pleiotrophin is administered in a concentration ranging from about 5 xcexcg to about 1 mg/ml of the defect area. In addition, tricalcium phosphate, hydroxyapatite, gypsum, or other suitable physiological mineral sources can be combined with the compositions to assist in repair of damaged or diseased bone. In accordance with an embodiment, a physiological compatible mineral comprises up to 80% of the bioactive mix of the present composition. Alternatively, the physiological compatible mineral may comprises about 5% to about 50% of the bioactive mix, and more preferably comprises about 5% to 30% of the bioactive mix. In addition, the present compositions can be combined with known pharmaceuticals and bioactive agents to create a delivery system for the local treatment of bone disorders or diseases.
In addition, the bioactive component of the present compositions can be further combined with growth factors, growth factor binding proteins, or eukaryotic cells. Examples of suitable growth factors comprise: fibroblast growth factor, transforming growth factor (e.g., TGF- ), bone morphogenetic protein, epidermal growth factor or platelet-derived growth factor. Examples of growth factor binding proteins are insulin-like growth factor binding proteins (IGFBP""s) such as IGFBP 3 and 5. Examples of suitable eukaryotic cells comprise bone marrow cells, osteoblasts, and mesenchymal stem cells. The bioactive composition of the present invention can further include an osteogenic agent that stimulates or accelerates generation of bone upon implantation into a bone defect site. Examples of osteogenic agents comprise demineralized bone powder, morselized cancellous bone, aspirated bone marrow, bone or cartilage forming cells, and other bone sources.
The bioactive compositions of the present invention are useful for stimulating the growth of bone and cartilage tissues at a predetermined localized site in a warm-blooded vertebrate. The method comprises contacting the site in need of repair with a composition comprising substantially pure pleiotrophin. In one embodiment the composition is surgically implanted at the site in need of repair and the composition comprises pleiotrophin and a polymer matrix, wherein the polymer matrix controls the release of pleiotrophin and concentrates pleiotrophin at the desired site. Alternatively, the composition may be in an injectable form and the method of contacting the site in need of repair comprises injecting the composition into or adjacent to the site. The injectable form of the present composition typically comprises pleiotrophin in combination with a pharmaceutically acceptable carrier. The viscosity of the compositions can be adjusted by controlling the water content of the compositions or by the addition of pharmaceutically acceptable fillers or thickening agents known to those skilled in the art. In one embodiment, the injectable forms include collagen fibers and the viscosity of the composition is controlled by adjusting the pH of the composition from about 6.0 to about 7.5.
The compositions of the present invention can be combined with an effective amount of antibiotics, chemotherapeutic agents, additional growth factors, antigens, antibodies, enzymes, or hormones. For example, a composition comprising pleiotrophin and an antibiotic may be useful in the treatment of osteomyelitis, thereby reducing the need for and risk of parenteral antibiotics. In addition, a composition comprising pleiotrophin and an antineoplastic agent could be used for the local treatment of bone neoplasm, or a composition comprising pleiotrophin and an osteogenic or other growth factor (e.g., osteogenin, bone morphogenetic protein, parathyroid hormone, or TGF- ) could be used to accelerate the repair of skeletal defects as occurs with excessive trauma and with skeletal deficiency disorders such as osteogenesis imperfecta.
As noted above the present compositions can be prepared in fluid forms for injection into a warm-blooded vertebrate. In one embodiment the injectable forms are used systemically to treat a warm-blooded vertebrate and provide therapeutic value for conditions such as arthritis or other pathogenic situations that involve bone and/or cartilage. The injectable pharmaceutical formulation may be administered via the parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in an isotonic saline, 5% glucose, or other well-known pharmaceutically acceptable liquid carrier. In one embodiment the injectable composition comprises pleiotrophin and a pharmaceutically acceptable carrier and the composition is administered intravenously.
In a preferred embodiment, the pleiotrophin compound is dissolved in a saline solution containing 5% of dimethyl sulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents are well-known to those familiar with the art and can be utilized as pharmaceutical excipients for delivery of the pleiotrophin compounds. Other delivery vehicles are contemplated for use in accordance with the present invention and can be used to administer the fluid forms of the present invention systemically to a warm-blooded vertebrate. For example the delivery vehicle may be an oral dosage form, an epidermal patch or other delivery vehicle known to those skilled in the art.